Dynamics of oxidative DNA demethylation from duplex DNA to chromatin

About This Grant

Project Summary/Abstract Methylation of cytosine bases in DNA to 5-methylcytosine (5-mC) acts as a key regulator in gene expression for vertebrate animals and is associated with DNA packaging into heterochromatin. DNA demethylation is initiated by ten-eleven translocation (TET) methylcytosine dioxygenases like TET2, which oxidizes 5-mC, marking it for removed by thymine DNA glycosylase (TDG), which is then replaced by an unmethylated C by base excision repair. It remains poorly understood how the presence of chromatin impacts the efficiency of active DNA demethylation, although both TET2 and TDG interact with nucleosome core particles. Preliminary studies from our lab have shown that TDG diffuses on DNA to search for modified bases, and encountering nucleosomes can interrupt this search. Further, TDG was also demonstrated to interact directly with TET2. Based on these findings, we hypothesize that TET2 and TDG act together and utilize NCPs as a scaffold to efficiently remove methylated cytosines wrapped within. The experiments to test this hypothesis will be performed in two phases. In the mentored K99 phase, the candidate will be trained in several new techniques, including how to design and create knock-in cell lines with CRISPR/Cas9, perform single-particle tracking within live cells, reconstitute nucleosome core particles, and generate high-resolution structures with cryo-electron microscopy. This training will occur during AIM 1 where the candidate will determine the dynamics of TDG diffusing and binding chromatin in living cells, as well as binding oxidized 5-methylcytosine on a defined DNA tether in a single-molecule approach. Further, the structure of TDG on a nucleosome will be determined using cryo-EM. Each training goal will be performed with respective experts in the field and will be complemented with professional development courses at Pitt. In the independent R00 phase, the candidate will capitalize on these new skillsets gained during AIM 1: in AIM 2, the candidate will use innovative techniques to generate site-specific base modifications at defined genomic positions and chromatin states. Then, repair dynamics of TDG will be tracked via single-particle tracking of live cells. In AIM 3, the skillset gained in AIM1 for TDG will be applied to TET2, where the live-cell single-molecule dynamics of TET2 will be determined on duplex DNA and chromatin, on endogenous levels of 5-mC and induced higher levels of DNA methylation. These results will be complemented with studies of TET2 on long DNA tethers with defined substrates. Finally, in AIM 4, the candidate will utilize the training in generating reconstituted nucleosomes to test how TET2 and TDG cooperate on chromatin or on duplex DNA with enzymological, structural, and single-molecule approaches. These experiments will provide mechanistic insights into how TET2 and TDG work together to achieve oxidative demethylation in the context of chromatin. The training components of this project will supply the candidate with the skillset and background to establish the basis of his independent work in his future academic career.